Antigen

Antigen Class

TFs and others

Antigen

Nrf1

Cell type

Cell type Class

Neural

Cell type

Brain

MeSH Description

The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM.

Sample

source_name

brain

strain

C57BL/6

developmental stage

Postnatal Day 1

genotype

wild type

antibody

NRF1 (Abcam, Cat# ab55744)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

The whole brains were homogenized with a glass douncing homogenizer. The cell homogenate was fixed with 1% paraformaldehyde for 10 min and quenched with 0.125 M glycine for 5 min at RT, followed by twice wash with PBS. Cell pellets from above steps were lysed in LB1 buffer (50 mM HEPES, pH 7.5 at 4°C, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X; 10 min at 4°C), LB2 buffer (10 mM Tris, pH 8 at 4°C, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; 10 min at 4°C), and then resuspended in LB3 buffer (10 mM Tris, pH 7.5 at 4°C, 1 mM EDTA, 0.5 mM EGTA, and 0.5% N-Lauroylsarcosine sodium salt). Chromatin was sheared using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Dynabeads, chromatin was incubated with indicated antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with BSA-blocked protein G Dynabeads for 2 hr at 4°C. Immunoprecipitated DNA was eluted from the beads, reversed crosslinked at 65°C overnight, digested with protease K and RNaseA and then purified for library construction. Immunoprecipitated DNA (∼1-30 ng) was end-repaired using End-It Repair Kit, tailed with deoxyadenine using Klenow exo-, and ligated to custom adapters with T4 Rapid DNA Ligase (Enzymatics). Fragments of 200-600 bp were size-selected using Agencourt AMPure XP beads (0.5X and 0.3X), and subjected to PCR amplification using Q5 DNA polymerase. Libraries were size-selected using Agencourt AMPure XP beads (0.75X), quantified by Qubit™ dsDNA HS Assay Kit and quality checked by High Sensitivity D1000 ScreenTape. Libraries were sequenced as 50 bp single-end reads on the Illumina HiSeq 4000 platform.

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

33396240

Reads aligned (%)

55.6

Duplicates removed (%)

39.7

Number of peaks

620 (qval < 1E-05)

mm9

Number of total reads

33396240

Reads aligned (%)

55.5

Duplicates removed (%)

39.8

Number of peaks

613 (qval < 1E-05)